Protein kinases are a family of enzymes that catalyse the phosphorylation of hydroxyl groups in proteins. Approximately 2% of the genes encoded by the human genome are predicted to encode protein kinases. The phosphorylation of specific tyrosine, serine, or threonine residues on a target protein can dramatically alter its function in several ways including activating or inhibiting enzymatic activity, creating or blocking binding sites for other proteins, altering subcellular localisation or controlling protein stability. Consequently, protein kinases are pivotal in the regulation of a wide variety of cellular processes, including metabolism, proliferation, differentiation and survival (Hunter, T. Cell, 1995, 80, 224-236). Of the many different cellular functions known to require the actions of protein kinases, some represent targets for therapeutic intervention for certain disease (Cohen, P. Nature Rev. Drug Disc., 2002, 1,309-315).
It is known that several diseases arise from, or involve, aberrant protein kinase activity. In humans, protein tyrosine kinases are known to have a significant role in the development of many diseases including diabetes, cancer and have also been linked to a wide variety of congenital syndromes (Robertson, S. C. Trends Genet. 2000, 16, 265-271). Serine/threonine kinases also represent a class of enzymes, inhibitors of which are likely to have relevance to the treatment of cancer, diabetes and a variety of inflammatory disorders (Adams, J. L. et al. Prog. Med. Chem. 2001, 38, 1-60).
One of the principal mechanisms by which cellular regulation is affected is through the transduction of extracellular signals across the membrane that in turn modulate biochemical pathways within the cell. Protein phosphorylation represents one course by which intracellular signals are propagated from molecule to molecule resulting finally in cellular responses. These signal transduction cascades are regulated and often overlapping as evidenced by the existence of many protein kinases as well as phosphatases. It is currently believed that a number of disease and/or disorders are a result of either aberrant activation or inhibition in the molecular components of kinase cascades.
Three potential mechanisms for inhibition of protein kinases have been identified thus far. These include a pseudo-substrate mechanism, an adenine mimetic mechanism and the locking of the enzyme into an inactive conformation by using surfaces other than the active site (Taylor, S. S. Curr. Opin. Chem. Biol. 1997, 1, 219-226). The majority of inhibitors identified/designed to date act at the ATP-binding site. Such ATP-competitive inhibitors have demonstrated selectivity by virtue of their ability to target the more poorly conserved areas of the ATP-binding site (Wang, Z. et al. Structure 1998, 6, 1117-1128).
There exists a need for the provision of further compounds that are inhibitors of protein kinases.
MAPKAP-K2 (mitogen-activated protein kinase-activated protein kinase 2) is a serine/threonine kinase that operates immediately downstream of the p38 kinase in the stress-induced MAPK pathway (FIG. 1).
The p38 kinase pathway is involved in transducing the effects of a variety of stress-related extracellular stimuli such as heat shock, UV light, bacterial lipopolysaccharide, and pro-inflammatory cytokines. Activation of this pathway results in the phosphorylation of transcription and initiation factors, and affects cell division, apoptosis, invasiveness of cultured cells and the inflammatory response (Martin-Blanco, Bioessays 22, 637-645 (2000)).
p38 kinase itself activates a number of protein kinases other than the MAPKAP kinases such as Mnk1/2, PRAK and MSK1 (FIG. 1). The specific and/or overlapping functions of the majority of these targets have yet to be resolved. This pathway has been of particular interest for the discovery of new anti-inflammatory agents. Previous strategies to intervene this pathway have involved the development of selective inhibitors of p38 kinase. Such inhibitors are effective both for inhibiting pro-inflammatory cytokine production in cell-based models and animal models of chronic inflammations (Lee et al., Immunopharmacology 47, 185-201 (2000)). p38 kinase knockout mouse is embryonic lethal. And cells derived from such embryos have demonstrated a number of abnormalities in fundamental cell responses. These observations indicate that caution should be paid to the long-term therapy with p38 kinase inhibitors.
An alternative strategy for the development of anti-inflammatory agents could be the inhibition of this pathway at the level of MAPKAP-K2. Human MAPKAP-K2 has two proline-rich domains at its N-terminus followed by the kinase domain and the C-terminal regulatory domain. This kinase has low homology with other serine/threonine kinases except MAPKAP-K3 and -K4. The C-terminal regulatory domain contains a bipartite nuclear localisation signal and a nuclear export signal. The crystal structure of inactive MAPKAP-K2 has been resolved (Meng, W. et al. J. Biol. Chem. 277, 37401-37405 (2002)). Activation of MAPKAP-K2 by p38 kinase occurs via selective phosphorylation of threonine residues 222 and 334 (Stokoe et al., EMBO J. 11, 3985-3994 (1992)). MAPKAP-K2 has an amphiphilic A-helix motif located within its C-terminal region that is likely to block the binding of substrates. The dual phosphorylation by p38 kinase has been proposed to reposition this motif resulting in enhanced catalytic activity (You-Li et al., J. Biol. Chem. 270, 202-206 (1995)). MAPKAP-K2 is present in the nucleus of unstimulated cells, and translocates to the cytoplasm upon cell stimulation. This kinase is known to phosphorylate a number of nuclear transcription factors as well as cytosolic proteins such as heat shock proteins and 5-lipoxygenase (Stokoe et al., FEBS Lett. 313, 307-313 (1992), Werz, et al., Proc. Natl. Acad. Sci. USA 97, 5261-5266 (2000), Heidenreich, et al., J. Biol. Chem. 274, 14434-14443 (1999), Tan, et al., EMBO J. 15, 4629-4642 (1996), Neufeld, J. Biol. Chem. 275, 20239-20242 (2000)). All such substrates contain a unique amino acid motif (XX-Hyd-XRXXSXX, where Hyd is a bulky hydrophobic residue) that is required for efficient phosphorylation by MAPKAP-K2 (Stokoe et al., Biochem. J. 296, 843-849 (1993)).
Currently MAPKAP-K2 is the only p38 kinase substrate for which a specific function has been identified. A specific role for MAPKAP-K2 in mediating the inflammatory response has been strongly indicated by the phenotype of the MAPKAP-K2-deficient mouse (MAPKAP-K2−/−) (Kotlyarov, et al., Nature Cell Biol. 1, 94-97 (1999)). This mouse is viable and normal except for a significantly reduced inflammatory response. Recently it has also been shown that MAPKAP-K2 deficiency results in a marked neuroprotection from ischaemic brain injury (Wang et al., J. Biol Chem. 277, 43968-43972 (2002)). MAPKAP-K2 is believed to regulate the translation and/or stability of important pro-inflammatory cytokine mRNAs. It is thought to function via phosphorylation of proteins that bind to the AU-rich elements found within untranslated regions of these cytokines. The identity of these proteins is currently under investigation.
MAPKAP-K2 therefore represents an intervention point in the stress-induced kinase cascade for perturbation of the inflammatory response.